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Use and Interpretation of Serologic and Skin Tests in the Respiratory Mycoses: Current Censidereflens" Charlotte C. Campbell, B.S.
It has been known for more than two decades that coccidioidomycosis and histoplasmosis occur predominantly as primary pulmonary infections from which most persons recover spontaneously without specific therapy, and indeed, without realizing they have experienced infection with a mycotic agent. The devastating disseminated diseases by which both mycoses were first recognized are seen in relatively few individuals. There are, of course, all stages of infection and disease between these extremes which are manifested in protean and doubtless still unrecognized ways. The development and use of skin and serologic tests were highly instrumental in effecting the current clinical and epidemiologic concepts of both these mycoses. These immunologic tools have been much less satisfactory or lacking for the study of blastomycosis and cryptococcosis which impressive and cumulative evidence suggest have a similarly broad spectrum, both clinically and epidemiologically. The purpose of this report is: (1) to summarize for the clinician the current status of serology in the diagnosis and follow-up of coccidioidomycosis, histoplasmosis, blastomycosis and cryptococcosis, and (2) to discuss certain related factors that may be helpful in the use and interpretation of both skin and serologic tests. SEROLOGIC TESTS
Coccidioidomycosis: Complement fixation (CF) and tube precipitin (TP) tests as described by Smith and eo-workers'P are still the time-honored procedures. Coccidioidin is used as antigen in both. Both types of test are necessary since the TP test becomes positive within one to three weeks after - From the Departments of Microbiology and Tropical Public Health, Harvard School of Public Health, Boston, Massachusetts. Investigations of the author and her associates cited in this report were supported in part by grants from the New York Tuberculosis and Health Association, The American Thoracic Society, the u. S. Public Health Service Grant 5 TI AI 177 for the Harvard Training Program in Tropical Medicine, and Research Grant (NIAID) 1 ROI AI07520.
onset of mild to moderately severe primary infections, in a large percentage of which CF tests never become positive. Additionally, precipitins are inconstantly transitory and in many cases no longer demonstrable in as little as four to six weeks. Serologic tests requested too late in the course of CF negative patients may thus be negative in persons actually recovering from coccidioidomycosis. Antibody demonstrable by the CF test, on the other hand, usually develops more slowly, and in those patients in whom it does appear, is not only diagnostic but prognostic. Patients with CF levels of 1:16-1:32 should be observed closely, clinically and serologically, for evidence of pulmonary or extrapulmonary dissemination. 'The higher the CF titer, the poorer the prognosis" has become almost axiomatic in assessing the extent and severity of both acute and chronic coccidioidomycosis. Conversely, decrease in CF level connotes an improved clinical status. There are exceptions to this, Le., central nervous system disease, in which lower or even negative CF titers do not foreshadow the usually grave prognosis. Generally, however, change in level of CF antibody is an invaluable clue to the patient's changing clinical status. Within the past five years an immunodiffusion (ID) test has been devised by Huppert and Bailey.s,. More recently a latex particle agglutination (LPA) test has been developed by Hyland Laboratories." The ID test correlates well in detecting CF reactive sera, while results of the LPA test correspond with those of the TP test. These four tests were evaluated, singly and in combinations, for capacity to detect cases ultimately diagnosed as coccidioidomycosis. The investigators found that no one test was adequate, but that the ID-LPA combination detected 93 per cent and the CF-TP combination 85 per cent of the patients diagnosed as having coccidioidomycosis." The ID and LPA tests provide an excellent combination for screening sera. The procedures are -Los Angeles, California.
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technically simple and rapid. Results are available in four minutes for the LPA test and usually within 24 hours for the ID test when the specimen is positive. In addition, all the necessary equipment and reagents, including positive and negative serum controls are Commercially available from Hyland Laboratories, While these tests are useful for screening sera from suspected cases of coccidioidomycosis, a small number of false positive reactions do occur. Furthermore, the tests provide no information as to the extent of infection or disease. For these reasons, Huppert and associates" have emphasized that sera from all positive reactors in the screening tests be forwarded to one of the Central Laboratories routinely performing CF tests. Histoplasmosis: Complement fixation using two types of antigen-histoplasmin and a saline suspension of Histoplasma capsulatum yeasts (WYP )-is again the time-honored procedure. Neither type of antigen reacts in all cases," In primary pulmonary infections CF antibody to WYP antigen is generally demonstrable within ten to 21 days after exposure to the organism, or frequently by the time symptoms appear. If there is a critical level indicative of dissemination, as in coccidioidomycosis, this has not been determined. Levels as high as 1:256 to 1:2048 have been observed within several weeks after onset of severe but uncomplicated spontaneously healing primary infections which decrease gradually to negative or very low levels (1:8-1:16), usually within four to six months. The harbinger of impending difficulty in histoplasmosis is the "plateauing" of CF titers, usually at 1:32 or above, over a period of several months instead of the expected steady decline to negative. This pattern or a fluctuating one, Le., from negative « 1:8) to "positive" (1:8 or » to negative and back to "positive" within several months, is highly suggestive of chronic extrapulmonary or pulmonary, often cavitary histoplasmosis. Antibody demonstrable with histoplasmin develops later in the primary pulmonary case and titers are considerably lower than with the WYP antigen. Conversely, histoplasmin reacts to significantly high titers in sera from certain chronic cases in which those with the WYP antigen are low or negative." As in coccidioidomycosis, CF antibody is never demonstrable with either antigen in certain of the milder primary cases. To detect these, as well as to find a test simpler than the CF, many other types of serologic tests have been tried." In nearly all, histoplasmin is used as antigen. The two most universally accepted are the Histoplasmin Latex Agglutination test (HLA), commercially available
50
from Consolidated Laboratories, Inc.," and the ID test" for which standardized reagents are not yet commercially available. The HLA test is satisfactory for detecting the primary pulmonary case. However, in a preliminary investigation this test was not reactive in CF positive sera from approximately 50 per cent of cases known to be chronically active," The lILA test thus is least reliable in detecting patients most needful of medical assistance and therapy. There is considerable controversy over interpretation of the ID test." Until this is resolved, demonstration of one or more precipitin linesm and/ or h or others-s-is considered a "positive" test. However, it is to be re-emphasized that all sera reactive in ID or HLA tests should be forwarded to a central laboratory for the CF tests, as well as those negative in the IU..A from patients in whom the clinician's index of suspicion of histoplasmosis remains high. Blastomycosis: CF tests, using a yeast phase antigen of Blastomyces dermatitidis, have been used as extensively as those for coccidioidomycosis and histoplasmosis. However, a summary of results in over 100 culturally confirmed cases revealed as early as 1960 that tests were positive in less than 40 per cent. 6 In a more recent assessment of ID tests using blastomycin, results were similarly discouraging.7 The Blastomyces antigens currently used for both skin and serologic tests are seriously lacking in specificity for antibody to this fungus and at the same time are highly reactive in many persons with active histoplasmosis or coccidioidomycosis. 8, 'I ,10 The antigens continue to be used in serologic tests because of their greater sensitivity rather than their poorer specificity, and thus often provide the Brst confirmation that one is dealing with a mycosis even if not blastomycosis, per see Cryptococcosis: Serologic tests for this mycosis have not been widely employed. The three which show the greatest promise are the indirect fluorescent antibody test, 11 bentonite particle agglutination test 12 and the latex particle agglutination test. 1S This latter has been adapted to detect excess antigen in body fluids, which may be important in the immunologic delineation of this and other mycotic diseases, as well as circulating antibody. None of these tests, however, has be~n developed beyond the earliest stages. They have been tried, even collectively, in very few cases compared with the tests for the three other mycoses. Little more than that they are available for further trial can be said. CryptococcaI antigens for skin tests are in an even earlier stage of development. ·Chicago Heights, Illinois.
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F ACI'ORS COMPLICATING INTERPRETATION OF SIaN AND SEROLOGIC TESTS
CWhat i8 a diagnostic and! or significant level antibody?'~ This is the question asked most frequently by clinicians. Unfortunately, there is no simple answer. As stated many times and illustrated by the foregoing, the Significance of antibody at any level varies with the clinical type, stage and extent of infection or disease and the sensitivity and specificity of the antigen. The levels of "positivity" arbitrarily chosen for epidemiologic or case-finding surveys have little place in interpreting serologic results in individual cases. The Significance ( interpretation) of the "positive" test (Le., 1:8 or greater for Histoplasma and Blastomyces antigens in the CF or HLA agglutination test; 1:2 or greater for the coccidioidin CF; and the demonstration of one or more precipitin lines in ID tests with any of the antigens) can be determined only in subsequent serologic tests in which the contemporary clinical state of the patient, the possibility of cross reaction and of antibody stimulation by diagnostic skin tests, the geographic distribution of the causative fungi in soils-and man's own movement of and across these soils-must all be considered. Cross reactions: Cross reactions of Histoplasma antigens in coccidioidomycosis sera were observed by Smith'" as early as 1940. They have since been repeatedly demonstrated not only in coccidioidomycosis, but in blastomycosls.v'" Additionally, it has been found that antigens of B. dermatitidis are much more cross reactive in histoplasmosis and coccidioidomycosis than specifically reactive in blastomycosis. 6 ,7,l o The coccidioidins used in CF tests, on the other hand, have proved to be amazingly specific.1 ,2 ,6 Those used in the ID and LPA tests appear to be reasonably specific at the present time, but have not been as extensively employed in other mycoses as the coccidioidins used in the CF.8,5 Cross reactions of blastomycins and histoplasmins used in ID or HLA tests have been little investigated. However, there is little reason to suppose that they are more specific than those used in the CF, for they are prepared in the same way and frequently are the same preparations. (N.B. It is the antigen that is specific or nonspecific or sensitive or insensitive, not the type of serologic procedure in which it is used. ) The patterns of cross reactivity are fully described in an earlier report." Briefly, they are summarized as follows: Histoplasma and Blastomyces antigens react in the CF test with sera from primary pulmonary coccidioidomycosis, sometimes to
of
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titers as high as 1:64-1:128, in which CF antibody to coccidioidin is later in developing or never develops. This cross can be especially misleading to clinicians outside endemic areas for coccidioidomycosis. It is essential to rule out recent travel in those areas or exposure to materials originating in those areas (fomitic infections) in patients who are skin-test positive to coccidioidin and to obtain serial serum specimens for TP-ID-LPA and further CF tests. This cross is rarely observed in late primary, chronic pulmonary or chronic disseminated coccidioidomycosis. Nor is it troublesome in primary cases when CF titers with all three antigens are equal or separated by only several tube dilutions. The greater specificity of coccidioidin is indicative that the infection is due to Coccidoides immitis. Cross reactions between Histoplasma and Blastomyces antigens seldom can be similarly resolved. In primary pulmonary histoplasmosis, it is 1J8tUJl for CF titers with Blastomyces antigens to equal those with Histoplasma antigens; and not unusual for antibody to Blastomyces antigens to be demonstrable earlier, react to higher levels and persist longer than to Histoplasma antigens. Few cases of primary pulmonary blastomycosis have been observed and the reactivity of the two antigens is not known for this type case. Both antigens react in sera from chronic cases of both diseases in seemingly haphazard, still undefined ways. Experience has revealed, however, that "the antigen yielding the higher titer" is an unreliable criterion for diHerentiating the specific etiology of these two diseases even in patients followed serologically over several years. Again by experience, the wiser course in acute pulmonary infections in which the two antigens equally react is to consider both histoplasmosis and coccidioidomycosis over blastomycosis; and in chronic cases histoplasmosis over blastomycosis, leaving establishment of specific etiology to intensified cultural, histologic or other procedures. Effect of skin tests on serologic results: Interpretation of serologic results has been further complicated by the belated confirmation that a single diagnostic skin test with histoplasmin can indeed stimulate production of circulating antibody in a certain percentage of healthy individuals hypersensitive to this antigen. The percentage varies from relatively low (10 per cent) to relatively high (> 50 per cent) in the separate studies, us doubtless reflecting natural differences in the populations tested and! or differences in the lots of antigens used in both skin and serologic tests. There is general agreement, however, that circulating antibody
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308 is not thus stimulated in histoplasmin skin test
negative persons. There is also general agreement that antibody "boosts" from the skin test are most readily demonstrated by the antigen histoplasmin, the same type of preparation used in the skin test. The "boost" has been observed in all types of serologic tests-CF, ID, HLA and others-using this type of antigen. Occasional "boosts" in the CF test have also been reported with WYP antigens, but these are much rarer than with the histoplasmins, and the elevations are minimal. Perhaps even more disturbing is the recent finding that a single skin test with coccidioidin may also stimulate circulating CF antibody demonstrable by both types of Histoplasma antigens in coccidioidin skin test positive persons, even though the serologic tests-CF, TP and ID-with coccidioidin remained negative.!" Thus, single coccidioidin or histoplasmin skin tests may enhance the level of circulating antibody to Histoplasma antigens in healthy persons hypersensitive to histoplasmin and! or coccidioidin. Similarly critical studies have not been done with Blastomyces antigens. One of the first reports on the controversial issue of skin tests es possible antibody "boosts," however, revealed that multiple applications of the histoplasmin skin test stimulated antibody to Blastomyces antigens in the CF test. 17 In view of the close reciprocal antigenic relationship of these two organisms, this is not surprising. Elevation in antibody resulting from a single skin test with one or more of the "ins" usually is not high. In the vast majority of hypersensitive persons, CF titers are increased from negative « 1:8) to 1:8, 1:16 or 1:32. In occasional persons, however, antibody levels may reach 1:64-1:256. The response is demonstrable in as little as five days after application of the test( s) and persists in some persons for as long as six months. The maximum CF levels, moreover, occur within two to three weeks after skin test( s) or within precisely the same time interval antibody levels increase in naturally acquired histoplasmosis. Antibody stimulated by the skin test( s) is even more readily demonstrated in ID tests with histoplasmin, appearing as the m precipitin line. Although, hopefully, it is now widely recognized that fungus pathogens are not exempt from these two very usual immunologic phenomena (cross reaction and stimulation of antibody by additional antigen) such observations do not negate the value of either skin or serologic tests. On the contrary, they have led to a more enlightened use of the tests, an improved evaluation of both in reaching specific diagnosis, and to an awareness that the here52
tofore recommended practice of skin testing with the three preparations simultaneously may also require reassessment. Nothing is known of a possible cumulative stimulatory effect. For these reasons, it is now advised that a base line serum be drawn before application of the skin test(s ) or, where this is not practical, instantly one or more becomes positive, usually within 24-4B hours. In individual cases, it may also be necessary to weigh whether the more useful information can be derived from skin or serologic tests, and where feasible withhold the skin tests completely until several serial serologic determinations have been done-except where there is a high index of suspicion of coccidioidomycosis outside endemic areas. In this respect, it is important to consider that hundreds of thousands of healthy persons have a marked dermal hypersensitivity to histoplasmin and! or coccidioidin, and that positive reactions to the fungal antigens do not carry the same public health connotations as those to old tuberculin (O.T.) or purified protein derivative (PPD), despite the clinical similarity and long historic association of the respiratory mycoses and tuberculosis. Endemic areas: Cryptococcus neoformans and Histoplasma capsulatum are essentially worldwide in distribution in small-pockets of soil contaminated with accumulations of bird, particularly pigeon, and bird and! or bat excreta, respectively. Both organisms apparently can persist in such microfoci, possibly others, in macroclimates that vary from tropical to temperate with a wide range in mean annual temperature and precipitation.P Coccidioides immitis, on the other hand, appears to be limited to microfoci in arid soils and the macroclimate peculiar to the southwestern United States, much of Mexico and other isolated areas in Central and South America.!" Blastomyces dermatitidis has been recovered from soils in very few instances. Knowledge of the distribution of this organism is scant and derives principally from the reporting of autochthonous cases which recently have been described from South African nations and several Central and South American countries.IO,IS Hence, except for coccidioidomycosis, history of residence or travel as aid in diagnosis of mycotic disease is more and more meaningless. This is due not only to the greater mobility of populations over the extensive areas known to be endemic for several of the species, but to man's own increasing upheavals of these foci in soils as he constructs or reconstructs highways, suburbs and old urban centers in communities with comparatively stable populations.!" The latter aspect which
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receives so much attention in arid coccidioidomycosis areas has been little emphasized as a means of disseminating the other respiratory mycotic agents. However, the outbreaks of histoplasmosis in Montreal.P Greenwood, South Carolina.t! and Mason City, Iowa22 involving construction activities in the communities were particularly instructive not only in this respect, but in again pointing up the deceptiveness of low prevalence of histoplasmin hypersensitivity as an index of whether H. capsulatum is endemic to an area or not. Too frequently this means only that the microfoci have not been disturbed, either recently or never before. Further, in an age of almost continuous construction, these three well-documented outbreaks forewarn that it is unwise to exclude histoplasmosis (or cryptococcosis or blastomycosis for which skin test antigens are lacking or less satisfactory) because of absence of travel to known endemic areas or identifiable exposure to classic constricted point sources as chicken houses, bird roosts or bat caves. The more recent evidence suggests that, as populations congregate, the high rates of histoplasmosis infections and hypersensitivity arise-as in coccidioidomycosis-from an activity in a community of which the affected individuals are unaware or accept as commonplace in contemporary life; and that following the disturbance of a single focus, these rates may change in any community, including one's own, from low to high literally overnight. 22 Variations in antigens and the general unavailability of the tests: Despite almost 20 years of study, the mycotic antigens are still difficult to reproduce consistently from lot to lot and continue to be the subject of intensive research by a number of investigators. The American Thoracic Society in particular deserves much credit for sponsoring a committee as well as research to attack the problems of mycotic antigens as well as 'tuberculins'.28 Variations in antigens, however, are the principal reasons why serologic tests are retained within the province of relatively few centralized laboratories" in which personnel are experienced in preparing °National Communicable Disease Center (NCDC), Atlanta, Ga., (Dr. Kaufman) and Kansas City Kansas (Dr. Tosh); New York State Dept. of Health, Xibany, (Dr. Gordon), and approximately ten of the state health laboratories ill the mown highly endemic areas for histoplasmosis and coccidioidomycosis. Veterans Administration Hospitals in Nashville, Tenn., (Dr. McDearman), Jackson, Miss., (Dr. Busey) and San Fernando, California, (Dr. Huppert); Walter Reed Army Institute of Research~ Washington, D. C., (Mr. Fife); and on a consultative basis only the University of California, Davis, (Dr. Pappagianis), University of Kentucky, Lexington, (Dr. Fureolow), Harvard School of Public Health, Boston, (Miss Campbell) and the Hektoen Institute, Chicago, (Miss McMillen).
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and standardizing these amazingly complex substances or have access to standardized preparation of others. The tests, per se, are among the simplest laboratory procedures. Even the more complex CF test is performed in many laboratories with other than mycotic antigens. The situation has improved within the very recent past. As noted earlier, consistently satisfactory reagents are now commercially available for ID and LPA screening tests for coccidioidomycosis and HLA screening tests for histoplasmosis, if used as recommended by the developers. In addition, histoplasmin containing both hand m components is available upon request from the Biological Reagents Section, NCDC, Atlanta, for' screenmg ID tests for histoplasmosis. Each of the four antigens used for CF tests is also available upon request from NCDC to laboratories performing a 50 per cent end point CF procedure routinely. The problems with preparing and standardizing skin test antigens are not as complex and are considerably less pressing. For example, a satisfactory lot of skin test antigen that would be sufficient for a million skin tests (perhaps over ten to 15 years) is exhausted in a matter of months by the centralized laboratories routinely performing the serologic tests on a large scale. It was thus possible for many private investigators to carry out extensive skin test surveys with a single lot of antigens and for the skin test preparations to be made available much earlier commercially." Additionally, skin test antigens are standardized to measure a single parameter-dennal hypersensitivity-whereas serologic antigens must be capable of demonstrating antibody produced at different stages of extremely protean diseases. Until the multiplicity of components in these complex, usually dimorphic, agents can be more consistently reproduced for ultimate isolation and characterization, commercial production ofserologic antigensexcept for screening tests-is not advocated. It is clear, however, that production of vaccines, as well as improved antigens for skin and serologic tests, must be more seriously considered in the future. Unlike many other types of infectious diseases, the mycotic diseases-respiratory and otherappear to become more prominent as man progresses, industrially and medically. Improvement of the very socioeconomic conditions so relevant to man's health in other spheres is irrelevant to the soil nurtured fungi as man increasingly disturbs "Coccidioidin (Cutter Laboratories, California); histoplasmin and blastomycin (Parke, Davis Co.). Histoplasmin tine tests have recently been licensed and are available from Lederle Laboratories.
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them, converting them into air pollutants.P Since neither man's activities nor the fungi in nature can be easily controlled, vaccination appears to be the only answer. In this respect, it is important to consider that the mycoses are new to contemporary medicine and that neither the morbidity nor mortality they produce has been even remotely assessed. SUMMARY
The foregoing is an effort to summarize for the clinician the current status of skin and serologic tests in the diagnosis of the respiratory mycoses, and to discuss certain factors which ongoing experience reveals are relevant to their use and interpretation, Le., specificity and sensitivity of antigens; stage and extent of infection or disease; cross reactions; antibody stimulation by skin tests; the extensive distribution of the fungi in nature and man's increasing disturbance of them lessening the value of history of residence and travel; and, finally, what serologic tests are available commercially or as Public Health Service contributions and where they are performed. ACKNOWLEDGMENT: The author wishes to express her gratitude for the generosity of the late Charles E. Smith, not only with his sera from coccidioidomycosis cases and coccidioidins, but, more importantly, his exuberant and stimulating ideas throughout almost 20 years of a most rewarding and happy association. Secondly, she wishes to apologize to the many whose significant contributions to this rapidly developing field have not been cited specifically in the interest of brevity. For these, see Reference 7. REFERENCES
1
2 3
4
5
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C. E., SAITO, M. T., BEARD, R. R., KEpp, R. M., CLARK, R. W., AND EDDIE. B. U.: Serological tests in the diagnosis and prognosis of coccidioidomycosis, Amer. I. HVg., 52:1, 1950. SMITH, C. E., SAITO, M. T., AND SIMONS, S. A.: Pattern of 39,500 serologic tests in coccidioidomycosis, I.A.M.A., 160:546, 1956. HUPPERT, M., AND BAlLEY, J. W.: The use of immunodiffusion tests in coccidioidomycosis. I. The accuracy and reproducibility of the immunodiffusion test which correlates with complement fixation, Amer. I. Clin. Path., 44:364, 1965. IDEM: The use of immunodiffusion rests in coccidioidomycosis. II. An immunodiffusion test as a substitute for the tube precipitin test, Amer. I. Clin. Path., 44:369, 1965. HUPPERT, M., PETERSON, E. T., SUN, S. H., CHITJIAN, P., AND DERREVERE, W.: Evaluation of a latex particle agglutination test for coccidioidomycosis, Amer. ]. Clin. Path., 49:96, 1968. SMITH,
6 CAMPBELL, C. C.: The accuracy of serologic methods in diagnosis, Ann. N.Y. Acad. Sci., 89:163, 1000. 7 CAMPBELL, C. C.: Serology in the respiratory mycoses. A review, Saboureudia, 5:240, 1967. 8 HEIl\'El\, D. C.: Diagnosis of histoplasmosis using precipitin reactions in agar gel, Pediatrics, 22:616, 1958. 9 HILL, C. B., AND CAUPBELL, C. C.: Commercially available histoplasmin sensitized latex particles in an agglutination test for histoplasmosis, Mycopathologia et Mycologia Applicata, 18:169, 1962. 10 BUSEY, J. F., AND HINTON, P. F.: Precipitins in blastomycosis, Amer. Rev. Resp. o«, 95:112,1967. 11 VOGEL, R. A.: The indirect FA test for the detection of antibody in human cryptococcal disease, I. Inf. Dis., 116:573, 1966. 12 KIMBALL, H. R., HASENCLEVER, H. F., AND WOLFF, S. M.: Detection of circulating antibody in human cryptococcosis by means of the bentonite flocculation test, Amer. Rev. Resp. Dis., 95:631, 1967. 13 CORDON, M. A., AND VEDDER, D. K.: Serologic tests in diagnosis and prognosis of cryptococcosis, I.A.M.A., 197: 961, 1966. 14 S~nTH, C. E.: Histoplasmin sensitivity and coccidioidal infection. I. Occurrence of cross reactions, Amer. I. Pub. Health, 39:722, 1949. 15 KAUFMAN, L., TERRY, R. T., SCHUBERT, J. H., AND McLAUGHLIN, D.: Effects of a single histoplasmin skin test on the serological diagnosis of histoplasmosis, I. Bact., 94:798, 1967. 16 PAPPAGIANlS, D., SMITH, C. E., AND CAMPBELL, C. C.: Serologic status after positive coccidioidin skin reactions, Amer. Rev. Resp. Dis., 96:520, 1967. 17 SASLAW, S., AND CAMPBELL, C. C.: Effect of histoplasmin skin testing on serologic results, Proc. Soc. Exp. Btol. and Med., 82:689, 1953. 18 AJELLO, L.: Comparative ecology of respiratory mycotic disease agents, Bact. Rev., 31(1):6, 1967. 19 CAMPBELL, C. C.: Histoplasmosis and other respiratory mycoses in the tropics. Industry and Trap. Health, VI: (Library Congo No. 52-34882) 145, 1967. 20 LEZNOFF, A., FRANK, H., TELNER, P., ROSENSWEIG, J., AND BRANDT, J. L.: Histoplasmosis in Montreal during the fall of 1963, with observations on erythema multiforme, Canad. Med. Assoc. 1.,91:1154,1964. 21 SELLERS, T. F., JR., PRICE, W. N., JR., AND NEWBERRY, W. M., JR.: An epidemic of Erythema multiforme and Erythema nodosum caused by histoplasmosis, Ann. Int. Med., 62:1244, 1964. 22 TOSH, F. E., DOTO, I. L., D'ALESSIO, D. J., MEDEmOS, A. A., HENDRICKS, S. L., AND CHIN, T. D.: A second of two epidemics of histoplasmosis resulting from work on the same starling roost, Am. Rev. Besn. Dis., 94:406, 1966. 23 PROCEEDING INT. CONF. ON MYCOBACTERIAL AND FuxGAL ANTIGENS, Am. Rev. Resp. Dis., 92:1,1965. Reprint requests: Miss Campbell, Department of Microbiology, Harvard School of Public Health, Boston, Massachusetts 02115.
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